Miguel A. De la Rosa

Laser flash kinetic analysis of Synechocystis PCC 6803 cytochrome c6 and plastocyanin oxidation by Photosystem I

Abstract Laser flash absorption spectroscopy has been used to investigate the kinetics of electron transfer from reduced cytochrome c 6 and plastocyanin to photooxidized P700 in Photosystem I (PS I) particles from the cyanobacterium Synechocystis PCC 6803. Data analysis yields second-order rate constants of 1.3 · 10 7 M −1 s −1 and 1.0 · 10 7 M −1 s −1 for the heme- and copper-proteins, respectively. With the two donor proteins, the observed rate constants ( k obs ) present a linear protein-concentration dependence, thus suggesting an apparent one-step bimolecular collisional mechanism. At neutral pH, the k obs values monotonically increase with increasing NaCl or MgCl 2 concentration, which is ascribed to the involvement of repulsive electrostatic interactions between the donor proteins and PS I. The difference in the effective concentration at which MgCl 2 has its maximum effect as compared with that of NaCl is attributed to the specific role played by Mg 2+ ions, which could act as electrostatic bridges between negatively charged groups. At physiological mild acid pH, cytochrome c 6 is a more efficient electron donor than plastocyanin. The inversion of the NaCl and MgCl 2 effect at pH below 5 — that is, decreasing of k obs with increasing ionic strength — is interpreted as arising from the involvement of attractive ionic interactions at pH lower than the isoelectric point of the donor proteins. Some evolutive aspects on the mechanism of electron donation to PS I are discussed.

Structure of the complex between plastocyanin and cytochrome f from the cyanobacterium Nostoc sp. PCC 7119 as determined by paramagnetic NMR. The balance between electrostatic and hydrophobic interactions within the transient complex determines the relative orientation of the two proteins.

The complex between cytochrome f and plastocyanin from the cyanobacterium Nostoc has been characterized by NMR spectroscopy. The binding constant is 16 mm–1, and the lifetime of the complex is much less than 10 ms. Intermolecular pseudo-contact shifts observed for the plastocyanin amide nuclei, caused by the heme iron, as well as the chemical-shift perturbation data were used as the sole experimental restraints to determine the orientation of plastocyanin relative to cytochrome f with a precision of 1.3 Å. The data show that the hydrophobic patch surrounding tyrosine 1 in cytochrome f docks the hydrophobic patch of plastocyanin. Charge complementarities are found between the rims of the respective recognition sites of cytochrome f and plastocyanin. Significant differences in the relative orientation of both proteins are found between this complex and those previously reported for plants and Phormidium, indicating that electrostatic and hydrophobic interactions are balanced differently in these complexes.

Photosynthesis: A new function for an old cytochrome?

In many cyanobacteria and algae, cytochrome c6 transports electrons between the cytochrome bf complex and photosystem I, replacing plastocyanin when copper is deficient. Higher plants, however, were thought to lack cytochrome c6 (refs 1,2) until the existence of a modified form in several species was inferred from genomic evidence. By measuring oxygen evolution with inside-out thylakoids, Gupta et al. inferred that heterologously expressed Arabidopsis cytochrome c6 can replace plastocyanin from Synechocystis or Arabidopsis in reconstitution experiments in vitro. From structural and kinetic evidence, however, we find that Arabidopsis cytochrome c6 cannot carry out the same function as Arabidopsis plastocyanin or as cytochrome c6 from the alga Monoraphidium braunii. This suggests that cytochrome c6 in higher plants may have lost its original primitive function in photosynthesis.

Nitration of tyrosine 74 prevents human cytochrome c to play a key role in apoptosis signaling by blocking caspase-9 activation

Tyrosine nitration is one of the most common post-transcriptional modifications of proteins, so affecting their structure and function. Human cytochrome c, with five tyrosine residues, is an excellent case study as it is a well-known protein playing a double physiological role in different cell compartments. On one hand, it acts as electron carrier within the mitochondrial respiratory electron transport chain, and on the other hand, it serves as a cytoplasmic apoptosis-triggering agent. In a previous paper, we reported the effect of nitration on physicochemical and kinetic features of monotyrosine cytochrome c mutants. Here, we analyse the nitration-induced changes in secondary structure, thermal stability, haem environment, alkaline transition and molecular dynamics of three of such monotyrosine mutants--the so-called h-Y67, h-Y74 and h-Y97--which have four tyrosines replaced by phenylalanines and just keep the tyrosine residue giving its number to the mutant. The resulting data, along with the functional analyses of the three mutants, indicate that it is the specific nitration of solvent-exposed Tyr74 which enhances the peroxidase activity and blocks the ability of Cc to activate caspase-9, thereby preventing the apoptosis signaling pathway.

Proteomic analyses of the response of cyanobacteria to different stress conditions

Cyanobacteria are significant contributors to global photosynthetic productivity, thus making it relevant to study how the different environmental stresses can alter their physiological activities. Here, we review the current research work on the response of cyanobacteria to different kinds of stress, mainly focusing on their response to metal stress as studied by using the modern proteomic tools. We also report a proteomic analysis of plastocyanin and cytochrome c 6 deletion mutants of the cyanobacterium Synechocystis sp. PCC 6803 grown under copper or iron deprivation, as compared to wild‐type cells, so as to get a further understanding of the metal homeostasis in cyanobacteria and their response to changing environmental conditions.

A comparative structural and functional analysis of cyanobacterial plastocyanin and cytochrome c 6 as alternative electron donors to Photosystem I

Plastocyanin and cytochrome c6 are two soluble metalloproteins that act as alternative electron carriers between the membrane-embedded complexes cytochromes b6f and Photosystem I. Despite plastocyanin and cytochrome c6 differing in the nature of their redox center (one is a copper protein, the other is a heme protein) and folding pattern (one is a β-barrel, the other consists of α-helices), they are exchangeable in green algae and cyanobacteria. In fact, the two proteins share a number of structural similarities that allow them to interact with the same membrane complexes in a similar way. The kinetic and thermodynamic analysis of Photosystem I reduction by plastocyanin and cytochrome c6 reveals that the same factors govern the reaction mechanism within the same organism, but differ from one another. In cyanobacteria, in particular, the electrostatic and hydrophobic interactions between Photosystem I and its electron donors have been analyzed using the wild-type protein species and site-directed mutants. A number of residues similarly conserved in the two proteins have been shown to be critical for the electron transfer reaction. Cytochrome c6 does contain two functional areas that are equivalent to those previously described in plastocyanin: one is a hydrophobic patch for electron transfer (site 1), and the other is an electrically charged area for complex formation (site 2). Each cyanobacterial protein contains just one arginyl residue, similarly located between sites 1 and 2, that is essential for the redox interaction with Photosystem I.

A single arginyl residue in plastocyanin and in cytochrome c(6) from the cyanobacterium Anabaena sp. PCC 7119 is required for efficient reduction of photosystem I.

Positively charged plastocyanin fromAnabaena sp. PCC 7119 was investigated by site-directed mutagenesis. The reactivity of its mutants toward photosystem I was analyzed by laser flash spectroscopy. Replacement of arginine at position 88, which is adjacent to the copper ligand His-87, by glutamine and, in particular, by glutamate makes plastocyanin reduce its availability for transferring electrons to photosystem I. Such a residue in the copper protein thus appears to be isofunctional with Arg-64 (which is close to the heme group) in cytochrome c 6 from Anabaena(Molina-Heredia, F. P., Dı́az-Quintana, A., Hervás, M., Navarro, J. A., and De la Rosa, M. A. (1999)J. Biol. Chem. 274, 33565–33570) andSynechocystis (De la Cerda, B., Dı́az-Quintana, A., Navarro, J. A., Hervás, M., and De la Rosa, M. A. (1999) J. Biol. Chem. 274, 13292–13297). Other mutations concern specific residues of plastocyanin either at its positively charged east face (D49K, H57A, H57E, K58A, K58E, Y83A, and Y83F) or at its north hydrophobic pole (L12A, K33A, and K33E). Mutations altering the surface electrostatic potential distribution allow the copper protein to modulate its kinetic efficiency: the more positively charged the interaction site, the higher the rate constant. Whereas replacement of Tyr-83 by either alanine or phenylalanine has no effect on the kinetics of photosystem I reduction, Leu-12 and Lys-33 are essential for the reactivity of plastocyanin.

Acetylsalicylic acid induces programmed cell death in Arabidopsis cell cultures

Acetylsalicylic acid (ASA), a derivative from the plant hormone salicylic acid (SA), is a commonly used drug that has a dual role in animal organisms as an anti-inflammatory and anticancer agent. It acts as an inhibitor of cyclooxygenases (COXs), which catalyze prostaglandins production. It is known that ASA serves as an apoptotic agent on cancer cells through the inhibition of the COX-2 enzyme. Here, we provide evidences that ASA also behaves as an agent inducing programmed cell death (PCD) in cell cultures of the model plant Arabidopsis thaliana, in a similar way than the well-established PCD-inducing agent H2O2, although the induction of PCD by ASA requires much lower inducer concentrations. Moreover, ASA is herein shown to be a more efficient PCD-inducing agent than salicylic acid. ASA treatment of Arabidopsis cells induces typical PCD-linked morphological and biochemical changes, namely cell shrinkage, nuclear DNA degradation, loss of mitochondrial membrane potential, cytochrome c release from mitochondria and induction of caspase-like activity. However, the ASA effect can be partially reverted by jasmonic acid. Taking together, these results reveal the existence of common features in ASA-induced animal apoptosis and plant PCD, and also suggest that there are similarities between the pathways of synthesis and function of prostanoid-like lipid mediators in animal and plant organisms.

Site-directed Mutagenesis of Cytochromec 6 from Synechocystis sp. PCC 6803 THE HEME PROTEIN POSSESSES A NEGATIVELY CHARGED AREA THAT MAY BE ISOFUNCTIONAL WITH THE ACIDIC PATCH OF PLASTOCYANIN

This paper reports the first site-directed mutagenesis analysis of any cytochrome c 6, a heme protein that performs the same function as the copper-protein plastocyanin in the electron transport chain of photosynthetic organisms. Photosystem I reduction by the mutants of cytochromec 6 from the cyanobacteriumSynechocystis sp. PCC 6803 has been studied by laser flash absorption spectroscopy. Their kinetic efficiency and thermodynamic properties have been compared with those of plastocyanin mutants from the same organism. Such a comparative study reveals that aspartates at positions 70 and 72 in cytochrome c 6 are located in an acidic patch that may be isofunctional with the well known “south-east” patch of plastocyanin. Calculations of surface electrostatic potential distribution in the mutants of cytochromec 6 and plastocyanin indicate that the changes in protein reactivity depend on the surface electrostatic potential pattern rather than on the net charge modification induced by mutagenesis. Phe-64, which is close to the heme group and may be the counterpart of Tyr-83 in plastocyanin, does not appear to be involved in the electron transfer to photosystem I. In contrast, Arg-67, which is at the edge of the cytochrome c 6 acidic area, seems to be crucial for the interaction with the reaction center.

Oxidizing Side of the Cyanobacterial Photosystem I EVIDENCE FOR INTERACTION BETWEEN THE ELECTRON DONOR PROTEINS AND A LUMINAL SURFACE HELIX OF THE PsaB SUBUNIT

Photosystem I (PSI) interacts with plastocyanin or cytochrome c 6 on the luminal side. To identify sites of interaction between plastocyanin/cytochromec 6 and the PSI core, site-directed mutations were generated in the luminal J loop of the PsaB protein fromSynechocystis sp. PCC 6803. The eight mutant strains differed in their photoautotrophic growth. Western blotting with subunit-specific antibodies indicated that the mutations affected the PSI level in the thylakoid membranes. PSI proteins could not be detected in the S600R/G601C/N602I, N609K/S610C/T611I, and M614I/G615C/W616A mutant membranes. The other mutant strains contained different levels of PSI proteins. Among the mutant strains that contained PSI proteins, the H595C/L596I, Q627H/L628C/I629S, and N638C/N639S mutants showed similar levels of PSI-mediated electron transfer activity when either cytochrome c 6 or an artificial electron donor was used. In contrast, cytochromec 6 could not function as an electron donor to the W622C/A623R mutant, even though the PSI activity mediated by an artificial electron donor was detected in this mutant. Thus, the W622C/A623R mutation affected the interaction of the PSI complex with cytochrome c 6. Biotin-maleimide modification of the mutant PSI complexes indicated that His-595, Trp-622, Leu-628, Tyr-632, and Asn-638 in wild-type PsaB may be exposed on the surface of the PSI complex. The results presented here demonstrate the role of an extramembrane loop of a PSI core protein in the interaction with soluble electron donor proteins.